RESUMEN
In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.
Asunto(s)
Adenovirus A Aviar/genética , Interleucina-2/genética , Melanoma Experimental/inmunología , Animales , Embrión de Pollo/virología , Clonación Molecular/métodos , Femenino , Humanos , Interleucina-2/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Plásmidos , Análisis de SupervivenciaRESUMEN
Recombinant CELO avian adenoviruses carrying green fluorescent protein (GFP) and and human interleukin-2 (IL-2) genes were obtained by homologous recombination in cell culture. The resultant recombinant CELO viruses are reproduced in chick embryos in the renal tubular and chorionic allantoic membrane cells. The ability of CELO vectors to transduce human and animal cells was studied in vitro (in cell cultures) and in vivo (in laboratory animals). GFP gene delivery and expression in recombinant CELO virus in tumors in C57BL/6 mice were for the first time demonstrated for B16 melanoma. Human IL-2 gene expression and protein accumulation in allantoic fluid of chick embryos infected with CELO-IL-2 vector were detected for the first time.
Asunto(s)
Aviadenovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Interleucina-2/genética , Proteínas Luminiscentes/genética , Alantoides/metabolismo , Alantoides/virología , Animales , Células Cultivadas/virología , Embrión de Pollo , Proteínas Fluorescentes Verdes , Interleucina-2/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/virología , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.
Asunto(s)
Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Adenovirus A Aviar/genética , Técnicas de Transferencia de Gen , Proteínas Recombinantes/genética , Fosfatasa Alcalina/sangre , Alantoides/enzimología , Alantoides/metabolismo , Alantoides/virología , Animales , Células Cultivadas/virología , Embrión de Pollo , Femenino , Adenovirus A Aviar/patogenicidad , Expresión Génica , Ingeniería Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Genoma Viral , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/metabolismo , Transducción GenéticaRESUMEN
The patterns of RNA from cells infected with avian adenovirus chicken embryo lethal orphan (CELO) virus were analyzed by Northern hybridization method. Early RNAs are specifically hybridized with several fragments of the Eco-RI restriction map: A (49-81%), B (0.2-20.3%), D (81-92.5%), and E fragments of the Bam-HI CELO DNA restriction map. Early RNAs were not hybridized with C (37.8-49%), G (31-37.8%), and E (20.3-31%) fragments of the Eco-RI DNA CELO restriction map. At least 19 types of virus-specific RNA molecules homologous to 4 different regions of the virus genome are produced in CELO-infected permissive cells. Several classes of virus-specific RNAs were identified in CELO virus-transformed cell strains.
Asunto(s)
Aviadenovirus/genética , Genoma Viral , Transcripción Genética , Animales , Northern Blotting , Células Cultivadas , Embrión de Pollo , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
The genomic region of the avian adenovirus FAV1 (CELO) encoding the precursor to virion structural protein VI (p VI) and the major capsid protein hexon has been sequenced. The 223-unit sequence of the CELO pVI protein has two potential Ad endoproteinase cleavage sites and a conserved C-terminal sequence including the Cys residue supposedly involved in endoproteinase activation. The CELO hexon gene sequence predicts a 942-residue protein (106.7 kDa). Multiple sequence alignment with other six known hexon protein sequences (human, bovine, murine, and avian) reveals high overall homology. The identity is highest in the regions corresponding to the pedestals which from the base of the hexon, and lowest in the regions corresponding to the loops which are exposed on the outer surface of the virion.
Asunto(s)
Adenoviridae/clasificación , Aviadenovirus/clasificación , Proteínas de la Cápside , Cápside/genética , Proteínas Estructurales Virales/genética , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Aviadenovirus/genética , Aves , Cápside/biosíntesis , Cápside/química , Bovinos , Secuencia Conservada , ADN Viral/química , ADN Viral/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Programas Informáticos , Proteínas Estructurales Virales/biosíntesis , Proteínas Estructurales Virales/químicaRESUMEN
A nondefective recombinant human adenovirus 5 (Ad5) carrying the gene encoding for the porcine rotavirus outer capsid protein VP7 in the E3 region of the Ad5 genome has been obtained. mRNA for VP7 in recombinant Ad5KV14/VP7 infected cells was demonstrated by means of RT-PCR. Radioimmunoprecipitation of cell extracts infected with Ad5KV14/VP7 on early (12 h p.i.) and late (24 h p.i.) stages of adenovirus infection with rabbit polyclonal antiserum raised to purified rotavirus virions revealed a recombinant protein possessing the same electrophoretic mobility, 37 kDa, as the native rotavirus K VP7. One-dimensional peptide mapping also demonstrated the identity of the recombinant and native VP7.
Asunto(s)
Adenoviridae/genética , Antígenos Virales , Proteínas de la Cápside , Cápside/genética , Regulación Viral de la Expresión Génica , Rotavirus/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Ensayo de Radioinmunoprecipitación , Recombinación GenéticaRESUMEN
A new system for hammerhead ribozyme (Rz) expression was examined in which fowl adenovirus type 1 (CELO) virus-associated RNA (CELO VA RNA) was used as a vector for the incorporation of Rz to target the mRNA of secreted alkaline phosphatase (SEAP) both in vitro and in vivo. The Rz gene was integrated into the CELO VA RNA between the internal promoter boxes A and B; apparently this did not interfere with its transcription. Rz integrated into CELO VA RNA and, lacking the viral sequences, exhibited the same activity in vitro, Consequently, CELO VA RNA sequences did not inhibit the integrated Rz activity in vitro. In vivo experiments were carried out with human 293 cells by co-transfection with plasmids containing Rz and SEAP. Inhibition of enzyme activity was 50% in 48 h. We conclude that CELO VA RNA may be used for effective expression of hammerhead Rz.
Asunto(s)
Aviadenovirus/genética , Vectores Genéticos , ARN Catalítico/genética , ARN Viral/genética , Fosfatasa Alcalina/genética , Aviadenovirus/enzimología , Secuencia de Bases , Línea Celular , Expresión Génica , Genes Virales , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/genética , ARN Viral/química , TransfecciónRESUMEN
A ribozyme was constructed of the catalytic domain of tobacco ringspot virus satellite RNA and flanking sequences complementary to the target, secreted alkaline phosphatase (SEAP) mRNA. The ribozyme specifically cleaved the substrate in vitro; compared with that constant temperature. The relationship between specific endoribonuclease activity of the ribozyme and Mg2+ concentration was shown. The ribozyme was active in 293 cell line which was cotransfected with plasmids carrying the SEAP gene under control of RSV LTR promoter and the ribozyme gene under early HCMV promoter: SEAP activity reduced by half.
Asunto(s)
Fosfatasa Alcalina/genética , ARN Catalítico/genética , ARN Mensajero/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Línea Celular , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Nepovirus/genética , Oligodesoxirribonucleótidos , Plásmidos , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , ARN Viral/genéticaRESUMEN
A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.
Asunto(s)
Adenovirus Humanos/genética , Fosfatasa Alcalina/genética , Vectores Genéticos , Placenta/metabolismo , Adenovirus Humanos/enzimología , Fosfatasa Alcalina/metabolismo , Células Cultivadas , Clonación Molecular , Humanos , Cinética , PlásmidosRESUMEN
The plasmid pH5KV14 containing the right end of the Ad5 viral genome (45-100 map units) with deleted nonessential E3 region (78.5-84 map units) has been constructed. A helper-independent recombinant virus Ad5KV14 has been obtained as a result of in vivo transformation of the line 293 human cells by the pH5KV14 plasmid DNA and an EcoRI-A DNA fragment of the virus Ad5 and subsequent recombination. A nondefective recombinant virus Ad5KV17 containing the Ad5 genome with the E3 region substituted for the plasmid DNA fragment coding for the kanamycin-resistance has been constructed by a similar procedure.
